Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacterial pathogens known. This bacterium can be readily aerosolized and utilized as a bioweapon. The morbidity and mortality of tularemia are significant and, given the infectious capability of Francisella, a major outbreak would readily overwhelm the ability of even the largest U.S. medical centers. Consequently, Francisella is classified as a category A bioweapon by the US government. Genes encoded on the Francisella pathogenicity island (FPI), are responsible for the virulence of this bacterium. The stringent starvation protein A (SspA), the macrophage growth locus protein A (MglA) and the pathogenicity island gene regulator (PigR) mediate activation of these genes and are therefore essential for the virulence of Francisella species that infect humans. MglA and PigR are unique to Francisella whereas SspA proteins are found in multiple bacteria. The Francisella SspA, however, is unusual in that it does not homodimerize but rather functions as a heterodimer with MglA. PigR is a putative DNA binding protein with a predicted winged-helix-turn-helix motif. How SspA-MglA and PigR mediate FPI activation is unknown, as is the underlying molecular mechanism that these proteins use to sense infection. The overarching goal of this proposal is the molecular dissection of these mechanisms through the study of these virulence factors in the human pathogenic Francisella tularensis tularensis and holartica subspecies. Early studies implicated the ?alarmone?, guanosine-tetraphosphate (ppGpp), as key for Francisella virulence. We recently showed that ppGpp binds directly to MglA-SspA and unveiled the molecular details of this interaction by solving the MglA-SspA-ppGpp complex structure. Further, we showed that ppGpp binding to MglA-SspA mediates high affinity binding of PigR to this heterodimer. In this revised proposal, we shall leverage our recent discoveries to dissect all components of the Francisella virulence regulatory system, including critically, the Francisella RNA polymerase (RNAP). Our central hypothesis is that F. tularensis employs a conceptually novel form of virulence activation involving a unique RNAP that contains the virulence activating complex MglA-SspA as a core constituent. This is supported by ChIP-seq studies and RNAP purifications from Francisella cells. We shall test our central hypothesis and complete the proposed objectives through two Specific Aims. Specific Aim 1: Elucidate the high resolution structure of (MglA-SspA)-ppGpp-PigR and identify inhibitors of ppGpp binding to MglA-SspA. Specific Aim 2: Determine the structure of Francisella RNAP complexes by cryo-EM. The successful completion of these studies will reveal a new paradigm in transcription regulation and enable the rational design of novel anti-Francisella-virulence therapeutics.